PROJECT SUMMARY Dental plaque represents one of the most complex microbial communities or biofilms known to afflict man. Oral biofilm-related diseases, e.g. dental carries, gingivitis, periodontitis, and candidosis, impact a large population of all age groups and continue to impose a huge economic burden due to the lack of effective therapies. The development of dental plaque begins with the attachment of early bacterial colonizers to the tooth enamel, generating an adhesive matrix that then attracts intermediate and late colonizers. Actinomyces spp. are key early colonizers that play a prominent role in biofilm development by virtue of their ability to directly interact not only with the tooth surface but also with a number of both early and intermediate colonizers. Therefore, our studies have focused on dissecting the adhesive properties, i.e. fimbriae and non-fimbrial proteins, dictating these interactions and the mechanism of their assembly on the surface of Actinomyces oris ? the most abundant Actinomyces in the human oral cavity. During the past grant period, we identified the major co-aggregation factor named CafA, which mediates A. oris interaction with oral streptococci. Remarkably, CafA is found at the tip of a distinct fimbrial structure made of the pilus shaft FimA, although cafA is not genetically linked to the type 2 fimbrial gene cluster fimB-fimA-srtC2. Significantly, we found that spatial positioning of the pilus tip adhesin CafA is essential for CafA-mediated bacterial coaggregation and this process requires the housekeeping sortase SrtA. We also discovered a small membrane protein named SafA, conserved in the Actinomycetales order, which is critical for SrtA membrane association. Investigations into the essential nature of srtA revealed the convergence of two conserved pathways, SrtA-catalyzed cell wall anchoring and LytR-CpsA-Psr (LCP)-mediated glycosylation, on the cell wall anchored glycoprotein GspA, itself critical for A. oris formation of mono- and multi- species biofilms and membrane integrity. Thus, we propose that SrtA is the fulcrum for molecular assembly on the cell surface of Actinomyces. Using biochemical, genetic electron microscopic, and structural approaches, we aim to test this central hypothesis by examining the mechanism of pilus hijacking and polymicrobial interactions mediated by the major co-aggregation factor CafA in A. oris, elucidating the mechanism of SrtA modulation of CafA spatial positioning and CafA-mediated coaggregation, and examining the glycosylation mechanism of the cell wall anchored GspA that contributes to biofilm formation and membrane integrity. The conservation of sortase-mediated surface assembly in Gram-positive bacteria and the utilization of srtA essentiality for inhibitor screens in other Gram-positive pathogens thus magnify the significance of our studies on how sortase SrtA modulates polymicrobial interactions via surface display of adhesive factors.